The advent of genomic technologies has yielded abundant datasets documenting protein-chromatin interactions genome-wide. Translating genomic data into physiological mechanisms remains extremely challenging. The prior funding period built upon our foundation of a defined cohort of GATA-1/GATA-2 binding sites discovered via ChIP-seq and their functional attributes. Since GATA-1 replaces GATA-2 at these sites upon erythropoiesis, which often alters transcription, we designated these GATA switch sites. We developed evidence for an extremely innovative scenario in which two GATA switch sites at a locus (Gata2), both essential for embryogenesis, non-redundantly control distinct aspects of hematopoiesis - hematopoietic stem cell (HSC) generation from hemogenic endothelial cells (+9.5 site) or myeloid progenitor function (-77 site). This is the only example we are aware of in which two cis-elements at a locus exert essential, but distinct, functions in a multi-tiered developmental hierarchy and the only example of a cis-element required to generate a stem cell. As +9.5 and -77 alterations underlie non-malignant and malignant hematopoietic pathologies, the studies extend beyond understanding how master regulators establish/maintain genetic networks and how genetic/epigenetic mechanisms underlie stem/progenitor cell generation and function. Aim 1: To elucidate how a single cis-element controls HSC generation. We hypothesize that the multimeric complex on the +9.5 composite element in hemogenic endothelium ensures GATA-2 levels to establish/maintain a genetic network that promotes hematopoiesis. We will test models to explain how GATA-2 expression in +9.5-/- AGM stimulates HSC genesis. We will test whether signals targeting GATA-2 control HSC genesis in a +9.5- dependent manner and will use CRISPR/Cas9 to dissect how +9.5 regulates Gata2. Aim 2: To determine how two GATA switch sites at the same locus control distinct sectors in the hematopoietic hierarchy. +9.5 and -77 functions differ considerably. Hematopoietic clusters emerge from -77-/-, but not +9.5-/-, Aorta Gonad Mesonephros (AGM), consistent with a unique +9.5 role in HSC genesis. We will test the hypothesis that the two cis-elements use different mechanisms to control distinct components of hematopoiesis. Aim 3: To leverage rules governing +9.5 site function to discover novel regulators of hematopoiesis. Prioritization of 798 +9.5-like composite elements revealed a site that controls an unstudied gene encoding Sterile Alpha Motif protein 14. As Samd14 harbors a functional +9.5-like site, is expressed in hematopoietic stem/progenitor cells, has attributes distinct from known regulators of hematopoiesis, its downregulation reduces myeloerythroid progenitors, and we linked its function to that of Stem Cell Factor-c-Kit, we hypothesize that Samd14 is an important regulator of hematopoiesis. We will test models to explain how Samd14 regulates c-Kit signaling and whether it controls hematopoiesis via modulating this axis. In aggregate, the proposed studies will forge new paradigms of considerable fundamental and translational importance.